PROJECT SUMMARY Nonalcoholic fatty liver disease (NAFLD) is the accumulation of fat in liver cells that advance to the more severe form called nonalcoholic steatohepatitis (NASH) characterized by inflammation, scarring (cirrhosis), liver failure and hepatocellular carcinoma, warranting liver transplant. Despite the profound economic and health impacts of NAFLD/NASH, little progress has been made in the management of patients with these severe clinical conditions. While weight loss and anti-diabetic medications are cornerstones of treatment, there is considerable interest in identifying more direct therapeutic interventions. Such developments are currently hampered by lack of an animal model with high physiologic fidelity to the human condition, particularly with respect to the development of fibrosis, immune evasion and metabolic syndrome features that define human NASH. To that end, we propose to utilize our state-of-the-art gene-editing platform to engineer Ossabaw swine with the most potent mutation associated with human NASH, PNPLA3I148M. We hypothesize that Ossabaw pigs carrying PNPLA3I148M alleles will develop histologically severe liver injury typical of human NASH along with metabolic syndrome, with atherogenic dietary challenge. Founder animals will be subject to routine clinical chemistry, lipidomic workups, and histopathological examinations sufficient to qualify fidelity of the model. Success in this endeavor would warrant a Phase II submission wherein the impact of the PNPLA3I148M mutation could be more extensively investigated, namely through more detailed imaging, histologic, cytokine and immune cell phenotyping. In addition to state-of-the art gene editing platform with expertise of Drs. Melkamu (Veterinary Physiology) and Carlson (Animal biotechnology) from Recombinetics, we have engaged as co-investigators, experts in clinical and pathophysiological aspects of NASH in human and in swine nutritional model (Drs. Naga Chalasani and Tiebing Liang from Indiana University) biochemical aspects of NASH , from Vanderbilt University). Drs. Gerry O'Sullivan, a local veterinary pathologist from the University of Minnesota, and Dr. Pierre Bedossa, a world-renown expert in scoring liver histopathology, will serve as consultants. A reliable large animal model of will have tremendous impact on industry and academic research to develop and test new drugs and novel therapeutic approaches to treat this disease. and expertise in (Dr. Charles Robb Flynn NASH